A generic light microscope comprises a sample plane, which a sample to be analyzed can be placed upon and at least one light source for emitting light in the direction of the sample plane. Light coming from the sample can then be detected by means of a detection device for capturing a sample image.
Structured illumination microscopy (SIM) is an established method for analyzing a sample with high resolution. In so doing, a sample is illuminated with structured illumination light. This can mean any light having a spatially variable intensity distribution across the cross-section of the beam. In particular, light having a period intensity distribution across the cross-section can be used, for example a line pattern with lines that are illuminated and lines that are not illuminated.
A generic optical arrangement that is incorporated into a beam path of the light microscope between the light source and the sample plane can be used for generating structured illumination. The generic optical arrangement comprises at least a first and a second optical assembly for providing structured illumination light from incident light.
The optical assemblies can for instance be gratings. Successive representations of the gratings are generated in the sample plane. Sample images of various grating orientations are captured, which can be used to calculate a sample image with extended resolution.
Said extended resolution represents a considerable advantage compared to the conventional capture of wide-field images without structured illumination. However, more time is required to capture an image with structured illumination. Therefore, it can be considered a fundamental objective to keep the time required for capturing an image low. In so doing, the instrument-based set-up should be realizable as efficiently as possible and be economical.
Known optical arrangements do not meet these requirements.
For example, it is known to use a single grating for the representation of various grating orientations on the sample, and to rotate said grating into various orientations. Moreover, an optical image field rotator can be used behind the grating, for example an Abbe-Koenig prism. It can be used to rotate the image and hence to achieve a rotation of the grating representation on the sample plane. However, the rotation of a grating or an image field rotator is relatively time-consuming. In addition, interfering reflections of diffracted light may potentially occur.
Alternatively, optical beam splitting may be used to generate the interfering beams; yet, this approach can be complicated in terms of required instruments and problematic with regard to stability.
Furthermore, it is known to provide superpositioned gratings with different orientations on a substrate. Light of a desired order of diffraction is guided to the sample, while other orders of diffraction are hidden. However, this results in a high loss of intensity.
Finally, a plurality of gratings with different orientations may also be provided. They are selected in succession by a motor-driven grating changer. But again, the time required to change between the gratings is high.
All in all, known optical arrangements are associated with a high lag time for changing between two grating orientations and/or high mechanical requirements, for instance with regard to a high positioning capability of movable optical components. In addition, undesirable losses of intensity of the illumination light may occur.